Normalization of the α‐1Na/K‐ATPase Signalosome Rescinds Epigenetic Modifications in NASH‐Related Hepatocellular Carcinoma

Abstract

OBJECTIVEHepatocellular carcinoma (HCC) is one of the most aggressive human cancers and is characterized by an acquisition of multiple abnormal phenotypes driven by genetic and epigenetic alterations. Epigenetic changes including DNA methylation and histone modification are associated with uncontrolled cell growth and proliferation, and even initiation and progression of HCC from chronic inflammation with or without fibrosis in the liver. Among others, non‐alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are two major risk factors as both of them may develop cirrhosis and hepatocellular carcinoma (HCC) if left untreated. Most of the existing clinical and experimental reports provide only a snapshot of abnormal histone modifications in HCC rather than their dynamic changes. This makes it difficult to elucidate the significance of these changes in the development of HCC. Therefore, the objective of the present study is to investigate the role of histone acetylation/methylation mediated alterations using in vitro cell line and high fat diet animal model of non‐alcoholic steatohepatitis (NASH)‐derived liver carcinogenesis.HypothesisMost of the existing clinical and experimental reports provide only a snapshot of abnormal histone modifications in HCC rather than their dynamic changes. Hence, the present study hypothesised that the histone acetylation/methylation disturbances mediated by the α‐1Na/K‐ATPase signalosome may help to elucidate the significance of these changes in the development of HCC.METHODSIn vitro studies were done using two human HCC cell lines, complemented with our in vivo murine models of NASH and NASH‐HCC. Experimental groups were exposed to pNaKtide, a selective α‐1 subunit signalling inhibitor. Acetylated/tri methylated H3K9 in cell lysates and liver homogenates were measured by ELISA, and their expression by confocal microscopy on immuno‐stained livers. Significant differences among groups were established at p<0.05 using ANOVA/t‐test.RESULTSEpigenetic changes in cell lines and liver malignancies correlated with morphological changes in NASH progression to malignancy. Acetylated and tri‐methylated H3K9 were significantly increased in untreated cells and livers when compared with pNaKtide treated cells and animals (p<0.05). Quantitative analysis of H3acetylK9 and H3tri‐methyl K9 proteins on confocal images from cells and livers confirmed our findings.CONCLUSIONSNormalization of the α‐1Na/K‐ATPase signalosome in human HCC cell lines and murine NASH‐HCC results epigenomic reprogramming.